Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin.

Roberto Chulluncuy,Attilio Fabbretti,Jose Nakamoto,Carlos Espiche,Pohl Milón

doi:10.3390/antibiotics5040038

Abstract

Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR) Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform.

Highlights

Bacterial pathogens account for 38% of human infections [1] and, because of their potential to develop antibiotic resistance, represent a severe threat to human health
IF3 binds across the 30S platform, interacts with several intersubunit bridges, and responds to conformational states of the small subunit (Figure 1) [17,19,20,28,29]
The structure of IF3 NTD consists of a globular α/β fold, constituted by a four-stranded b-sheet onto which an α-helix is packed [30]

Summary

Introduction

Bacterial pathogens account for 38% of human infections [1] and, because of their potential to develop antibiotic resistance, represent a severe threat to human health. The problem is of particular importance in underdeveloped countries, where the incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) mycobacteria and bacteria is rapidly increasing Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a devastating disease with higher incidence in underdeveloped countries than in their developed counterparts ([2] and references therein). Kanamycin and Streptomycin bind the decoding site (A-site) of the minor ribosomal subunit (30S) and inhibit protein synthesis mainly by causing misreading of the mRNA [5] or translocation inhibition (Kanamycin) [6,7]. The streptomycin-resistant strains contain hyper-accurate ribosomes [8]

Methods

Results

Conclusion